1
A. INTRODUCTION
RATIONALE
Streptococcus suis is a gram positive cocci that could transmit from
animals (mainly pigs) to human via ingestion behaviors of uncooked
foods and non-intact skin exposure during pig butchering and pork meat
processing. It could cause an endemic with a high mortality rate raging
from 12,8% to 27,9%. Patients with S. suis infection after discharge
could have severe complications sush as hearing loss (from 50% to
66,4%), vestibular disorders (22,7%), limb gangrene and amputation.
These complications have a low recovery rate, leading to a great burden
in healthcare system and economic system. In Vietnam, disease due to
S.suis is a topical issue in the field of infectious diseases because of
specific epidemiological factors and severe clinical consequences,
especially in case of inappropriate diagnosis and late treatment. This
disease ranked on the 6th position among 10 infectious diseases with the
highest incidence and mortality rate. Currently, the resistance of S.suis to
almost antibiotics used in farming results in a high risk of antimicrobial
resistance in human. Resistance genes and virulence related genes have
been studied in the world. In Vietnam, major studies are mainly from the
South. Until now, despite a widespread circulation, there has been no
study on comprehensive evaluation of clinical and laboratory
characteristics, prognostic and virulence factors, distribution of these
features by clinical syndromes of S.suis in Vietnam. Therefore, we
conduct this research for three following objectives:
1 Describe clinical and laboratory characteristics, clinical
syndromes in patients infected with Streptococcus suis.
2 Determine prognostic factors of mortality in patients infected with
Streptococcus suis.
3 Evaluate the antibiotic susceptibility and identify some resistance
genes, virulence genes of Streptococcus suis.

positive anaerobes with a spherical shape. It is transmitted via uncooked
ingestion and exposure through wounds in the skin while contact with
infected pigs in butchering and uncooked food processing. S.suis breach
the epithelial barriers, exist in blood then invade multiple organs and
cause inflammatory reactions in these organs. Moreover, S.suis is able to
cross the blood-brain barrier to invade the central nervous system,
leading to meningitis.

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Epidemiology of Streptococcus suis infection in human
The first case of S.suis infection in human was described in 1968 in
Denmark. Since then, significantly increasing numbers of human cases
have been reported in many countries including Netherlands, Belgium,
Denmark, England, France, Germany, Sweden, Canada. Asia regions
such as Hong Kong, Taiwan, China, Thailand and Vietnam notably have
a great proportion of 90.2%. S.suis infection could cause an endemic,
typically in China where observed two outbreaks in 1999 and 2005 with
huge numbers of infected cases and a high mortality rate. In Vietnam, a
case of S.suis infection was first reported in November 1996 with
meningitis manifestation. Since then, human cases infected by S.suis
have been reported in all three regions in Vietnam. According to General
Department of Preventive Medicine – Ministry of Health, until 2016, the
rate of S.suis infection in human was 0.007 cases per 100,000 persons
and this disease was at the 6 th rank among 10 infectious diseases with the
highest incidence and mortality rate.

3

Clinical syndromes of S. suis infection and prognostic factors

tetracycline, erythromycin; some strains are resistant to ceftriaxon and
fluoroquinolone. Resistance genes were also identified such as erm(B)
(erythromycin), cat (chloramphenicol), tet(M), tet(O), tet(L)
(tetracyclin). Virulence factors are predominant existence in type 2 S.
suis which transmits directly from animals to human. Many studies in
the world indicated three major virulence factors including: protein
muramidase-released protein (MRP) encoded by mrp gene, suilysin
encoded by sly, extracellular factor (EF) encoded by epf gene.

6

Technique of molecular biology and the applications in S. Suis
research
The discovery of novel gene sequencing technology makes a
significant breakthrough compared to Sanger technology in time, quality
and cost of sequencing process. Whole genome sequencing of S.suis
provides aid for determining serotypes, MLST (Multilocus sequence
typing), resistance genes and virulence related genes.
Chapter 2. MATERIAL AND METHOD
Study population

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1

Patients had co-infection with HIV, HCV, HBV
Patients refused to participate in the study at any time regardless of
study period.
Study design: Prospective study.
Sample size: Convenience sampling, the study will recruited all patients
who satisfied inclusion criteria meet in 2.1.1, and who were treated at
National hospital of Tropical diseases from May 2015 to May 2018.
Method
Patients with definitive diagnosis of S. suis infection will be
classified into 3 groups: (1) Meningitis, (2) Sepsis with meningitis and
(3) Septic shock. We will not only evaluate and analyse general
characteristics but we also compare features of clinical findings,
laboratory tests and prognostic factors, resistance genes, virulence genes
and serotype.
2.4.1. Study process
a) Objective
1:
Evaluate
general
clinical
characteristics, laboratory results and clinical
syndromes in patients infected with Streptococcus
suis
Clinical syndromes caused by human infection of S. suis: Based on
clinical manifestations and sample culture or PCR results, clinical
syndromes were divided into three following groups:
Meningitis: patients with signs and symptoms of meningitis + CSF
culture and/or PCR is positive with S. suis.
Sepsis with meningitis: patients with clinical manifestations of sepsis
and meningitis had positive result of S. suis in both blood and CSF

2 Death group: patients were died at the hospital or patients went home for
death because of treatment unresponsiveness or the severity of disease
progression.
We utilized univariate and multivariate regression analysis of
clinical characteristics, laboratory results, exposure history and
comorbidities between two groups to identify prognostic factors of
mortality.
Treatment outcome: Mortality rate, average duration of treatment,
sequelae (hearing loss, paralysis, mental issues, limb amputation due to
gangrene after discharge).
c) Objective 3: Evaluate antibiotic susceptibility patterns and identify
some resistance genes, virulence genes of Streptococcus suis

•

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6
We identified antibiotic sensitivity of S. suis by susceptibility
testing, MIC calculation with E-test technique (E-test strips
manufactured by Biomerieux-France), which is based on the guidance of
CLSI M100.
Identification of resistance genes, virulence genes, serotypes and
genotypes via MLST were based on analysis of S. suis genome after
performing novel sequencing method by Miseq machine, using primer
from standard kit of NextaraXT library manufactured by Illumia –
American. Results were checked, analysed with experts from Hanoi
Oxford University Clinical Research Unit (OUCRU).
– Evaluate antibiotic sensitivity of S. suis: Determine the sensitivity of

Campaign (2012)
– Criteria for clinical diagnosis of meningitis: Based on Principles and
Practice of Infectious Diseases (2013).
– Criteria for alcoholism: Based on AUDIT-C score of WHO (2001) and
developed by Frank (2008). Patients were diagnosed with alcoholism
when they had total score ≥ 4 in men and ≥ 3 in women.
– Technique for Streptococcus suis identification using Realtime PCR:
Use a pair of primers and a probe for cps2 gene to recognize S. suis
o Primers:
• cps2JF (5’- GGTTACTTGCTACTTTTGATGGAAATT-3’)
• cps2JR (5’-CGCACCTCTTTTATCTCTTCCAA-3’)
o Probe: (FAM-TCAAGAATCTGAGCTGCAAAAGTGTCAAATTGATAMRA). Bioresearch (American)
+ Machine: 7500 Fast Real-time PCR (Applied Biosystem – American)
+ A process of real-time PCR for cps2 gene (based on the process
performed at Laboratory Department in National Hospital of Tropical
Diseases and OUCRU in Hanoi
– Culture and identification technique of S. suis: Equipment: Blood
culture system Bactec 9050/9120: Bactec Dickinson, American and
some machines from England, Germany, France; Sheep blood agar,
chocolate agar (Oxoid, England). Bacteria identification was based on
Vitek 2 compact system from Biomeriux, France.
– Susceptibility testing and MIC identification with E-test: Use
equipment and antibiotic strips (E-test) manufactured by Bio merieux,
France. Interpretation and Report based on CLSI M100.
– Novel sequencing technology and data analysis using bioinformatics
sofware: Follow current procedures in National Hospital of Tropical
Diseases and Oxford University Clinical Research Unit.
6 Data collection: Data collection was carried out via designed case report
form (CRF).
7 Data analysis: Software SPSS 16.0, STAT with appropiate statistical

59
26.70
• Pig butchering
41
18.55
• Uncooked food processing
11
4.98
• Pig breeding
7
3.17
Ingestion exposure
46
20.81
• Eat “tiet canh”
41
18.55
• Eat other raw foods
5
2.26
Ingestion + Contact
12
5.43
Unknown
104
47.06
There were 117 patients (52.94%) with medical history of
exposure to source of infection. Eating “tiet canh” made the largest
contribution of 23.08%. There were 47.06% patients with unknown
history of exposure.

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55.00
The most common symptom in S.suis patients were fever, headache
and shivering.
Table 3.3. Symptoms by category of clinical syndrome
Meningiti
Meningitis with
Septic
p
s
sepsis
shock
Symptoms
n=102
n=79
n=40
(%)
(%)
(%)
102
79
40
Fever
(100)
(100)
(100)
63
53
35
Shivering

46
20
Vomiting
0.26
(64.71)
(58.23)
(50.00)
9
14
25

Hemorrhagic necrotic
rash
Refill >2s

n=102
(%)
97
(95.1)
68
(66.67)
1
(0.98)
6
(5.88)
26
(25.49)

Sepsis with
Septic shock
meningitis
n=79
n=40
(%)
(%)
60
14
(75.95)
(35.00)
48
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were hemorrhagic necrotic rash, refill > 2 seconds and cold extremities.

3
–
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Laboratory characteristics
Biochemistry results.


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Table 3.6. Biochemistry features
Total
Meningiti Sepsis with
Septic
p
s
meningitis
shock
(n=221)
Biochemistr
y result
Frequenc Frequency Frequenc Frequency
y (%)
y (%)
(%)
(%)
Creatinin ≥
5
16

(97.50)
(66.06)
60
55
37
152
ALT ≥ 37

500 (ng/l)
(43.14)
(59.49)
(92.5)
(57.92)
1
(Chi-squared test)
There were a marked decline in prothrombin time, fibrinogen and
noticeable increase in D-dimer, APTTs in the group of septic shock
compared to remaining groups.
4 Treatment outcome
– Mortality rate: The overall mortality rate was 14.03%. The mortality
rate in patients of septic shock was 60%. 35.26% patients were death
within the first 24 hours of admission.
– Sequelae when discharged: Proportion of hearing loss in patient
population was 44.8%.

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Prognostic factors of mortality
Table 3.8. Regression analysis of epidemiological factors related to
mortality
p
p

aOR 95% CI
s
value
value
Diarrhea
No
1
1
13.9
Yes
6.23 2.78


≥37
7.90 1.83 34.13 0.01 1.63
12.37
1

0.04

0.51

0.64

Patients with Creatinin ≥120 μmol/l and total bilirubin >17 UI/l had
a higher mortality risk than other patients (OR were 21.78 and 4.3;
respectively, and p
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Antibiotic resistance of S. suis
Susceptibility test

Chart 3.2. Antibiotic susceptibility profile of S. suis
Low susceptibility was observed in erythromycin, clindamycin
and tetracyline whereas susceptibility of tetracyline was 0%.
Susceptibility of remaining antibiotics (peniciline, ampiciline,
ceftriaxone, linezolid, levofloxacin and vancomycin) were 100%.
– Occurrence of resistance genes.

Chart 3.3. Frequency of resistance gene
The most common resistance genes were ant(6) with 78.75%, tet
(B) with 75% and erm(B) with 60%, followed by tet(O) with 28.75% and
aph (3) with 21.25%.

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Distribution of serotype

–

Distribution of serotype
Table 3.12. Distribution of serotype

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Septic
shock
n=15
(% )
13
(86.67)
0
(0.00)
2
(13.33)

Total
n=80
(% )
74
(92.50)
4
(5.00)
2
(2.50)

p

0.05

The predominant serotype was 02. The occurrence of serotype 1/2 was
only observed in patients with septic shock (with the proportion of 13.33%).

3

ST1
(81.25
(78.38)
(89.29)
(73.33)
)
9
2
1
6
ST665
(11.25
(16.22)
(7.14)
(6.67)
)
1
1
0
2
ST105
(2.70)
(3.57)
(0.00)
(2.50)
0
0
1
1
0.08

difference in distribution of sequence type among groups of clinical
syndrome. ST25 and ST28 were 2 novel types. There were 02 samples
with undefined sequence type.
4 Occurrence of virulence genes
– Distribution of virulence genes
Table 3.14. Distribution of virulence genes
Meningiti Sepsis with
Septic
Total
s
meningitis
shock
Virulence
p
gene
n=37
n=28
n=15
n=80
(%)
(%)
(%)
(%)
37
28
14
79
mrp
0.11
(100)

(67.86)
(60.00)
(75.00)
Major virulence genes in isolated samples were mrp, epf và sly.
There was no significant difference in distribution of virulence genes
among groups of clinical syndrome
Chapter 4. DISCUSSION
4.1. General characteristics of study population
.1.1. Characteristics of age, gender, and monthly distribution
– Age characteristic
Our study on 221 patients showed that the majority of patients was
in their middle age ranging from 40 to 60 years old, constituting 69.23%.
Median age of patients was 53 years old. This finding is consistent with
several previously published studies, as the median age of S. suis
infection was 54 years old in a study in China [4], and 52.9 years old in
another study in Thailand [57]. In Vietnam, a study on 101 patients
conducted by Ho Dang Trung Nghia at Hospital of Tropical Diseases at
Ho Chi Minh City also showed a similar result (a median age of 50 years
old) [21]. Our result confirms again that S. suis infection was common
among middle-aged population (over 40 years old), but rare among
young population (under 30 years old).
– Gender characteristic.
Our study participants were predominantly male (92%). There was
little difference in gender characteristic between our study and previous

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studies. Several meta-analysis indicated that a proportion of male gender

suis infection through consumption of undercooked pig-related food
products to minimize risk of S. suis infection. Strategies to raise public
awareness and to change raw and undercooked pork eating habits should
be intensified, particularly in the context of high incidence of S. suis
infection in Vietnam.
4.2. Clinical and laboratory characteristics and clinical syndromes of
S. suis infection in human.
4.2.1. Clinical syndromes.
Meningitis was the most common clinical syndrome among our
study population, with a prevalence of 46.1%, followed by sepsis with
meningitis (35.7%) and septic shock (18%). The distribution of clinical

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syndromes varies greatly among different countries and studies with
different designs and diagnostic methods. According to
Wangsomboonsiri (2008), meningitis accounted for 52%, followed by
sepsis (27%) and septic shock (12%). In China, septic shock had a
prevalence of 64% in an outbreak in Jiangsu and of 28.9% in another
outbreak in Sichuan. This reflects that pathogenic properties and
distribution of S. suis strains varies among different countries.
4.2.2. Clinical characteristics
a) Symptoms.
All of our patients had fever as the onset symptom, which is
consistent with the outbreak in Sichuan, China. Symptoms on admission
were significantly different between patients with meningitis and those
with septic shock. In patients with only meningitis and patients with sepsis
accompanied by meningitis, the most common symptoms were headache,



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common sign in patients with septic shock by S. suis. Hemorrhagic
necrotic rash in our study was dark purple, macular, confluent rash
which usually begins on outer ear, head, face, neck, then spreads to trunk
and extremities. This could suggest an important clinical evidence to
early diagnose septic shock by S. suis and provide physicians with a
basis for an appropriate management and plan to minimize mortality and
to improve treatment effectiveness in patients with S. suis infection.
4.2.4. Laboratory characteristics
a) Features of serum biochemistry results.
Patients with septic shock had significantly abnormal liver and
kidney function results compared to the remaining groups of patients.
More than 90% of septic shock patients had abnormal liver and kidney
function tests. Our results concur with previous findings in several
studies in Thailand and China. Hongjue Yu et al reported a proportion of
liver and renal failure of 90% and 59% in patients with septic shock,
respectively, which was higher than in patients without septic shock
(68% and 3%). In Thailand, a 2009 study by Fongcon also showed
similar results: patients with septic shock had elevated liver enzymes,
creatinine and total bilirubin level. Severe hepatic and renal injuries in
patients with septic shock correlates with their clinical scenarios. The
majority of our septic shock patients had oliguria or anuria, some of
whom required hemodialysis to replace renal functions.
b) Characteristics of blood coagulation.
Blood coagulation results were also considerably different among
three groups of patients. Patients with septic shock had a significantly
higher proportion of abnormal coagulation tests – namely prothrombine
time, fibrinogen and D-dimer – than remaining groups (meningitis and

specialized in infectious diseases, where most patients are in a severe
condition and beyond the treatment and care capability of provincial and
primary care hospitals. In addition, most patients are referred to national
specialized hospitals in their late stages of disease, which could contribute
to a higher mortality rate in these hospitals.
4.3.2. Complications of patients with S. suis infection.
a) Hearing loss.
Hearing loss prevalence was 44.8% in our study. This finding
correlates with several previous studies in Vietnam and in foreign
countries. Some research reported that hearing loss and vestibular
disorders are two most common complications of S. suis infection in
human. Hearing loss prevalence varies greatly among different
studies, ranging from 6% to 100%, with a mean prevalence of 39%.
In Vietnam, hearing loss had a prevalence of 66.4% and 27.3%,
respectively.
b) Extremity gangrene.
A rare complication in our study that has never been mentioned in
the literature was extremity gangrene by S. suis. In our study, this
complication was found in 6 patients, 4 of whom had a clinical scenario
of septic shock, but only 3 of whom required limb amputation with the
proportion of 1.36%. These three patients, after a period of recovery
from shock or improved clinical scenarios, began to develop cyanosis in
distal extremities, and then turn into dry gangrene in middle and distal
phalanges. In these patients, distal extremities could not be preserved,
and therefore, required amputation.
4.3.3. Prognostic factors of mortality
a) Clinical epidemiological factors
Our study illustrated a higher mortality rate in patients exposed
through food ingestion than in those exposed through farming,
butchering or trading. In a study in Thailand by Wangsomboonsiri,

logistic regression analysis, demonstrated that mortality rate in patients
with increased fibrinogen consumption (fibrinogen level
aminoglycosides.
4.5.2. Serotype of isolated strains
In our study, a majority of isolated strains were serotype 2,
constituting 92.25%. Serotype 14 and serotype 1/2 represented lower
proportions, 5% and 2.5%, and particularly, the latter was only presented
in septic shock patients. These findings are in good agreement with a
study by Nguyen Thi Hoang Mai et al in which serotype 2 was the
principal pathogen of meningitis caused by S. suis in adults in Southern
Vietnam, with 150 out of 151 patients infected with S. suis serotype 2 and

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the only other patient infected with S. suis serotype 24 [8]. The majority of
previous studies in foreign countries and in Vietnam reported that S. suis
infection in human is predominantly caused by serotype 2, along with
other less common serotypes 1, 4, 14, 16.
4.5.3. Sequence type (ST) classification in isolated strains
81.25% of isolated strains in our study were S. suis ST1, and 9
were S. suis ST665, which differs from ST1 only in the allele profile of
gki gene. ST1 were reported to be the primary pathogen in adults in Asia
(Vietnam, Thailand, Hongkong, Japan, China, …), in Europe (along with
ST20 in Netherlands) and in South America (Argentina), whereas in
North America, among three prevailing sequence types of S. suis
serotype 2, ST25 and ST28 were more common than ST1. These two
sequence types were identified in two strains in our study.
4.5.4. Virulence genes
Approximately all isolated strains carried simultaneously all of
three virulence genes, mrp, epf and sly, with respective proportions of

evolution.
4.5.6. Identification of two strains with undefined sequence types
It is exceptional that we identified two strains with undefined
sequence types in this study. We certified that our sequencing database
were of good quality after a thorough re-examination to ascertain that
those two samples were not mistakenly sequenced to cause
misidentification of sequence types. The genome of those two strains
shared a number of fair similarities to that of the reference strain BM407
and other strains in our study. Those two strains, one isolated from a
patient with septic shock and meningitis and the other isolated from a
patient with meningitis only, all lacked several sequences in regions from
500 to 600 kbps and from 1000 to 1100 kbps compared to the reference
strain BM407. We are planning to contact with the authority of MLST
database to update sequence types of those two strains.
CONCLUSION

–

–

–

–

1. Clinical and laboratory characteristics and clinical syndromes
of S. suis infection in human.
Our study recruited 221 patients with three clinical syndromes of S. suis
infection, namely meningitis, sepsis with meningitis and septic shock.
This disease was predominantly found in men (90%) and in middle-aged
people between 40 and 60 years old (69.23%).

Antibiotic resistance genes identified in our study were ant(6) (78.75%),
tet(B) (75%), tet(O) (28.75%) and erm(B) (60%).
The most common S. suis serotype was serotype 2 (92.5%), followed by
serotype 14 (5%) and serotype 1/2 (2.5%).
The most common sequence type was ST1 (81.25%), followed by
ST665 (11.25%) and ST105 (2.5%). There were two strains with
undefined sequence types.
Over 90% of isolated strains carried mrp, epf and sly virulence genes.
Two sequence types, namely ST25 and ST28, were identified in Vietnam
for the first time.

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